Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion

<div><p>Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers <i>Oct4</i> and <i>Nanog</i>. In addition, methylation of several imprinted genes except <i>Peg3</i> was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.</p></div

Degree of methacrylation as determined by TNBS assay.

<p>(A) Various volume percentages of methacrylic anhydride (0.25%, 1.25% and 20%) were analyzed to investigate the degree of methacrylation of the synthesized fish GelMA. (B) Comparison of a high degree of methacrylation (20% MA) according to the origin of gelatin (fish GelMA vs. porcine GelMA); there was no significant difference. The percentage of incorporated substitution was calculated by comparing the amount of remaining amino groups (-NH₂) in GelMA to that in pristine gelatin. Error bars represent standard deviations (SDs) of measurements performed on six samples.</p

Bisulfite genome sequencing analysis of imprinted genes.

<p>DNA methylation patterns of paternally (<i>H19</i> and <i>Igf2</i>), and maternally imprinted genes (<i>Peg1</i> and <i>Peg3</i>) in ESCs, pESCs, NSCs, pNSCs, ES-pNSC, and pES-NSC hybrid cells. Black and white circles represent methylated and unmethylated CpGs, respectively.</p

Farmer's reasons to engage in organic agriculture

The master thesis deals with farmer's reasons to engage in organic agriculture. The theoretical part is devoted to the topic of organic agriculture, its development in the Czech Republic and abroad, organic farming legislation and subsidy policies in the Czech Republic. Described methodology is a part of master thesis. The empirical part provides the readers with the presentation of eight individual organic farms in the regions of Hana and Beskids. On the grounds of empirical survey carried out through in-depth semi-structured interviews, the thesis reveals inner motivations that stimulate farmer's commitment to work in an environment-friendly way. Then, detailed description and interpretation of the codes resulting from open coding of the interviews are presented

Degradation characteristics of fish GelMA hydrogels.

<p>(A) Degradation profiles of fish GelMA hydrogels with various degrees of methacrylation (low, medium and high) and GelMA concentrations (5%, 10% and 15%) upon exposure to collagenase type II. (B) Comparison of fish and porcine GelMA hydrogels with high degree of methacrylation and 10% gel concentration. Error bars represent SDs of measurements performed on three samples. Representative cross-sectional SEM images of fish GelMA (C~F) and porcine GelMA (G~J) hydrogels reveal different gel morphologies after degradation with collagenase type II. (K) Pore size distribution of GelMA hydrogels (Pore size frequency obtained from 5 SEM images per condition).</p

Behavior of cells encapsulated in fish GelMA hydrogels.

<p>(A-F) NIH3T3 cells embedded in medium and high degree of methacrylation fish GelMA containing 5% (w/v) GelMA were stained using calcein-AM/ethidium homodimer at 3 h and 24 h after encapsulation to evaluate the cell viability compared to high degree of methacrylation porcine GelMA. (G-I) After 2 days of culture, cells proliferated and elongated in fish and porcine GelMA hydrogel. (J-L) Representative images of the cells stained with phalloidin for the actin filaments (green) and nuclei counterstained with DAPI (blue) at 72 h. (P) Viability of the encapsulated cells. (Q) Quantification of cell proliferation in GelMA hydrogel until 5 days. Cell proliferation rate was not significantly different at all condition.</p

Synthesis of fish gelatin methacryloyl (GelMA) and fabrication of photocrosslinked GelMA hydrogel.

<p>(A) Gelatin was reacted with methacrylic anhydride (MA) to introduce a methacryloyl substitution group on the reactive amine and hydroxyl groups of the amino acid residues. (B) GelMA photocrosslinking to form A hydrogel matrix under UV irradiation. The free radicals generated by the photoinitiator initiated chain polymerization with methacryloyl substitution. (C) Schematic of formation of patterned hydrogels using photolithography.</p

Identification of Putative Biomarkers for the Early Stage of Porcine Spermatogonial Stem Cells Using Next-Generation Sequencing

<div><p>To identify putative biomarkers of porcine spermatogonial stem cells (pSSCs), total RNA sequencing (RNA-seq) analysis was performed on 5- and 180-day-old porcine testes and on pSSC colonies that were established under low temperature culture conditions as reported previously. In total, 10,184 genes were selected using Cufflink software, followed by a logarithm and quantile normalization of the pairwise scatter plot. The correlation rates of pSSCs compared to 5- and 180-day-old testes were 0.869 and 0.529, respectively and that between 5- and 180-day-old testes was 0.580. Hierarchical clustering data revealed that gene expression patterns of pSSCs were similar to 5-day-old testis. By applying a differential expression filter of four fold or greater, 607 genes were identified between pSSCs and 5-day-old testis, and 2118 genes were identified between the 5- and 180-day-old testes. Among these differentially expressed genes, 293 genes were upregulated and 314 genes were downregulated in the 5-day-old testis compared to pSSCs, and 1106 genes were upregulated and 1012 genes were downregulated in the 180-day-old testis compared to the 5-day-old testis. The following genes upregulated in pSSCs compared to 5-day-old testes were selected for additional analysis: matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 1 (MMP1), glutathione peroxidase 1 (GPX1), chemokine receptor 1 (CCR1), insulin-like growth factor binding protein 3 (IGFBP3), CD14, CD209, and Kruppel-like factor 9 (KLF9). Expression levels of these genes were evaluated in pSSCs and in 5- and 180-day-old porcine testes. In addition, immunohistochemistry analysis confirmed their germ cell-specific expression in 5- and 180-day-old testes. These finding may not only be useful in facilitating the enrichment and sorting of porcine spermatogonia, but may also be useful in the study of the early stages of spermatogenic meiosis.</p></div

Quantitative RT-PCR analysis of imprinted gene expression.

<p>The expression profiles of paternal and maternal imprinted genes were analyzed by real-time RT-PCR. All data are normalized to <i>ACTB</i> expression and calibrated on the ESCs, whose gene expression was considered 1 for all genes. Error bars represent mean values ± SEM of three independent experiments. Student’s t-test: ***, p<0.001; **, p<0.01; *, p<0.05.</p

Generation of fusion hybrid cells between parthenogenetic and biparental cells.

<p><b>(A)</b> GFP fluorescence images of fusion between biparental ESCs and parthenogenetic neural stem cells (ES-pNSC), and between pESCs and biparental neural stem cells (pES-NSC) at day 3 after fusion (200 ×). <b>(B)</b> GFP fluorescence images of ES-pNSC and pES-NSC hybrids after FACS sorting (100 ×). <b>(C)</b> Representative tetraploid karyotype of the hybrid cells.</p